LETTERS TO THE EDITOR has been suggested that PIEZO1 is the likely origin of the A gain of function variant in PIEZO1 (E756del) and 4,6 Psickle channel. sickle cell disease PIEZO1 codes for a mechanically activated ion chan - nel, and is widely expressed throughout the body, includ - The novel PIEZO1 E756del variant was recently iden - ing in the red cell membrane. It is a large protein with 36 tified in about 30% of Africans, and associated with sig - transmembrane domains. 8 Gain of function variants in nificant red cell dehydration and protection against PIEZO1 have been shown to cause dehydrated heredi - 1 malaria. Erythrocytes from people with the E756del vari - tary stomatocytosis, 9 whereas loss of function variants ant were found to have reduced infection by Plasmodium cause congenital lymphedema. 10 The properties of falciparum in in vitro studies, corresponding with equiva - PIEZO1 also fit well with what is known about P 1 sickle lent findings in a mouse model. As such, it might be including that it is a mechanosensitive channel and a expected to be an important determinant of severity in non-specific cation conductance pathway. 6 sickle cell disease (SCD). We have studied the prevalence A recent study identified a gain of function variant in and significance of this PIEZO1 variant in 788 patients PIEZO1 (E756del), which is associated with malaria with SCD. Surprisingly, we found that it had little or no resistance and increased red cell dehydration, and which influence on red cell phenotype. was present in about 30% of Africans. 1 It might be All the complications of SCD arise from the polymer - expected that this polymorphic variant would be an ization of hemoglobin molecules incorporating a mutated important determinant of severity in SCD. We have, 2 β globin chain (HbS), βS (HBB, c.A20T, p.Glu7Val). The therefore, investigated the hypothesis that the E756del rate of HbS polymerization is very dependent on the PIEZO1 allele causes increased red cell dehydration in intracellular concentration of HbS, with the lag time SCD, and is, therefore, associated with a more severe before the onset of polymerization increasing with the form of the disease, including increased anemia and concentration of HbS raised to the power of 30. 3 Small hemolysis. differences in red cell hydration, causing changes in HbS Adults and children with either sickle cell anemia concentration therefore have a marked effect on the rate (HbSS) or HbSC disease were recruited from specialist of HbS polymerization and consequent hematologic and clinics at King’s College Hospital. Data used in this study clinical complications. 4 came from patients in two separate studies: a study of In SCD, red cell cation loss and dehydration is thought cation transport in SCD, and a study of genetic determi - to occur through three major transport pathways: K-Cl nants of severity in SCD. Many patients were recruited 4 cotransport (KCC), the Gardos channel, and P sickle . The into both studies allowing the data to be combined. The interaction of these channels is complicated but results in studies were approved by the UK National Research a net loss of cations from the red cell, with subsequent Ethics Committee (11/LO/0065, 13/NW/0141, loss of water, and increased hemoglobin concentration. 5 07/H0606/165, 12/LO/1610), and all patients gave writ - Whereas the molecular basis of the Gardos channel ten consent in accordance with the 1975 Declaration of (KCNN4) and KCC (KCC1, KCC3, KCC4) are known, Helsinki, as revised in 2008. Psickle is a pathophysiological entity, whose molecular Blood samples for DNA extraction and cation transport basis is unknown. 6 measurements were collected when patients attended Psickle is a channel present only in sickle cell erythro - routine clinic appointments. Clinical and laboratory data cytes, which is activated by deoxygenation. It is non- were averaged from steady-state measurements recorded selective, allowing the passage of ions and other small in the electronic patient record over an approximately molecules; in particular, it is responsible for the entry of 10-year period (2004-2013). 11,12 Mean hospitalization calcium into the deoxygenated sickle erythrocytes which rates were calculated over ten years (2004-2013), divid - causes the dehydration cascade. 7 Various proteins have ing an individual’s number of hematology admissions by been proposed as the basis for P sickle , although recently it the number of observed years. This was used as a surro - Table 1. Hematologic parameters compared across different Piezo1 genotypes in patients with sickle cell anemia. P values from ANOVA, adjusted for age and sex. E756del/ E756del E756del/WT WT/WT P (7/7) n=46 (7/8) n=161 (8/8) n=405 n mean n mean n mean Hb (g/ L) 22 83.6 61 83.3 146 86.5 0.293 MCHC (g/L) 22 337 61 335 146 333 0.593 Retics (%) 13 13.9 36 11.7 83 12.1 0.304 Neutrophils (x10 9/L) 22 5.4 61 4.8 143 5.1 0.448 Platelets (x10 9/L) 22 387 61 401 146 396 0.917 LDH 21 434 61 454 138 423 0.493 HbF% 12 7.0 30 6.3 71 7.0 0.580 Psickle 8 2.0 25 1.6 50 1.7 0.191 Admissions per year 23 0.49 66 0.80 156 0.71 0.517 P-values from ANOVA, adjusted for age and sex. WT: wild type; Hb: hemoglobin; MCHC: mean corpuscular hemoglobin concentration; Retics: reticulocytes; LDH: lactate + -1 dehydrogenase; HbF: fetal hemoglobin; n: number. P sickle units: mmol K (l cells.h) . haematologica 2019; 104:e 91 LETTERS TO THE EDITOR Table 2. Hematologic parameters compared across different Piezo1 genotypes in patients with sickle cell anemia. E756del/E756del E756del/WT WT/WT P (7/7) n=11 (7/8) n=65 (8/8) n=120 n mean n mean n mean Hb (g/ L) 9 114 45 116 78 116 0.917 MCHC (g/L) 9 345 45 345 78 350 0.109 Retics (%) 6 5.3 34 4.6 48 4.7 0.676 Neutrophils (x10 9/L) 9 4.5 45 4.1 78 3.9 0.525 Platelets (x10 9/L) 9 229 45 265 78 237 0.297 LDH 9 274 42 248 73 244 0.428 HbF % 4 3.1 27 2.1 35 1.5 0.146 Psickle 5 0.96 23 1.2 43 1.0 0.69 Admissions per year 9 0.59 40 0.26 72 0.32 0.149 P-values from ANOVA, adjusted for age and sex. WT: wild type; Hb: hemoglobin; MCHC: mean corpuscular hemoglobin concentration; Retics: reticulocytes; LDH: lactate + -1 dehydrogenase; HbF: fetal hemoglobin; n: number. P sickle units: mmol K (l cells.h) . gate marker of pain frequency. Activity of P sickle was similar to the heterozygote frequency of 36% (9 out of 25 defined as the deoxygenation-induced Cl –-insensitive K + individuals) reported by Ma et al .1 As anticipated, we also transport in the continued presence of clotrimazole and identified a significant number of patients who were was measured according to published protocols. 13 homozygous for the deletion. Based on Ma et al .’s paper, DNA samples from 788 patients with SCD of African we were expecting to find that those patients with and African-Caribbean origin were tested for the pres - E756del showed evidence of increased red cell dehydra - ence of the E756del polymorphism, associated with tion (higher MCHC, increased P sickle activity) with faster increased red cell dehydration. Adequate data on the rate HbS polymerization causing more anemia and hemolysis. of hospital admissions was available for a subset of 366 This was evidently not the case in either HbSS or HbSC adult patients. A 204bp amplicon was generated encom - disease. In addition, we found more than 20 patients passing rs572934641 (-/TCC) on Chromosome 16 (UCSC with HbSS who were homozygous for E756del (7/7 Dec2013 Hg38 assembly) using published primer repeats), and hematologically identical to patients with sequences. 1 The Reverse primer was modified with FAM the wild-type genotype (8/8 repeats), confirming that this fluorescent dye. Denatured PCR product was fragment PIEZO1 allele does not significantly affect red cell cation sized by capillary electrophoresis on an ABI Prism 3130 transport in SCD, and is not an important determinant of Automated Sequencer (Applied Biosystems, Foster City, severity in SCD. CA, USA) with a Genescan 500 ROX size standard The lack of effect of E756del in SCD may suggest that (Applied Biosystems). Allele calling was performed on this PIEZO1 deletion has a relatively small effect on resulting traces in Genemarker v.1.95 (Soft Genetics, cation permeability, 1 although surprisingly Ma showed State College, PA, USA). ANOVA (IBM SPSS 24, New marked changes in osmotic fragility and red cell morphol - York, NY, USA) was used to compare the different clini - ogy associated with this polymorphism. In particular, the cal, laboratory and cation transport measurements across increased cation loss caused by E756del may be insignif - the different PIEZO1 E756 genotypes. icant in the face of the much larger cation fluxes present The E756 deletion occurs within a sequence of triplet in abnormal sickle red cells. 13 Our study shows that the repeats, with eight triplet repeats occurring in the wild E756del is not linked to P sickle activity (oxygen sensitive, type, and seven with E756del. The overall allele frequen - non-selective ion and small molecule permeability path - cy of E756del was 20.1% in our sample of 788 patients, way) 14 but does not necessarily disprove the hypothesis although phenotypic information was not available for that the PIEZO1 protein mediates some or all of the all patients.